Protein-based therapeutics currently represent one in every four new drugs approved by the FDA and command a market in excess of $30 billion. The vast majority of therapeutic proteins require additional post-translational modifications to attain their full biological and therapeutic function. One of the most important of these modifications, N-linked protein glycosylation, is predicted to affect more than half of all eukaryotic protein species and is often essential for proper folding, pharmacokinetic stability, systemic half-life, immunogenicity and overall efficacy for a large number of human therapeutic proteins. Since most bacteria do not glycosylate their own proteins, expression of many therapeutically relevant glycoproteins, including antibodies, is relegated to mammalian cells. Unfortunately, mammalian cell culture suffers from a number of drawbacks including low volumetric productivity, product heterogeneity, high media cost, retroviral contamination and the relatively long time required to generate stable cell lines. Recently, however, it has been reported that the Campylobacter jejuni asparagine-linked (Nlinked) protein glycosylation system can be functionally transferred into Escherichia coli, giving the recipient E. coli cells the ability to glycosylate proteins. Although the bacterial N-glycan is structurally different from its eukaryotic counterparts, it stands to reason that such glyco-enabled E. coli could be engineered to perform sequential glycosylation reactions that mimic the early processing of N-glycans in humans and other higher mammals. Such an accomplishment would open the door for performing complex human-like protein glycosylation in bacteria. Thus, the long-term goal of this research project is to "humanize" the N-linked protein glycosylation process in E. coli for the routine production of authentic human glycoproteins in this simple host organism. Towards this goal, the objective of this particular application is to begin the early stages of "humanizing" the bacterial glycosylation system via glycan engineering (specific aim 1) and site-specific transfer of novel glycans onto target proteins (specific aim 2). The specific hypothesis behind the proposed research is that reconstitution of a eukaryotic N-glycosylation pathway in E. coli will result in N-glycoproteins with structurally homogeneous human-like glycans. These studies are significant because they should (i) provide the necessary genetic tools to help elucidate the crucial role of glycosylation in a myriad of biological phenomena and (ii) enable the biotechnological synthesis of novel glycoconjugates and potential immunostimulating agents for research, industrial and therapeutic applications. PUBLIC HEALTH RELEVANCE: N-linked glycosylation is predicted to affect more than half of all eukaryotic protein species and is often essential for proper folding, pharmacokinetic stability, systemic half-life, immunogenicity and overall efficacy for a large number of therapeutically relevant proteins (e.g., human erythropoietin). Since most bacteria do not glycosylate their own proteins, expression of many therapeutically relevant glycoproteins, including antibodies, is relegated to mammalian cells despite the fact that mammalian cell culture suffers from a number of well-documented drawbacks. Therefore, the focus of these studies is the development of "glyco-engineered" bacteria that are capable of producing authentic human glycoproteins.